title: Double in situ detection of sonic hedgehog mRNA and pMAPK protein in examining the cell proliferation signaling pathway in mouse embryo.
abstract: Double in situ detection of RNA molecules and proteins in tissue sections is not trivial. A successful experiment heavily depends on the preparation of the tissue as well as the quality of the probes and antibodies. Detection of two or more molecular markers also requires reagents and experimental conditions that will preserve authenticity (accuracy) of the single staining patterns. Here, we describe in detail the protocols used to detect sonic hedgehog (Shh) mRNA by in situ hybridization and immunofluorescence staining for phosphorylated mitogen-activated protein kinase (pMAPK) in the same mouse embryonic tissue sections. In addition to protocols for manual immuno-staining, we provide data from automated machine-based staining protocols and highly recommend it to achieve strong signal and reproducible results.
authors.affiliation: Developmental Biology Program, Molecular Cytology Core Facility, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
authors.forename: Sho
authors.initials: S
authors.lastname: Fujisawa
authors.forename: Mesruh
authors.initials: M
authors.lastname: Turkekul
authors.forename: Afsar
authors.initials: A
authors.lastname: Barlas
authors.forename: Ning
authors.initials: N
authors.lastname: Fan
authors.forename: Katia
authors.initials: K
authors.lastname: Manova
mesh_terms: D000818:Animals; D049109:Cell Proliferation; D004622:Embryo, Mammalian / Q000378:metabolism / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D053823:Hedgehog Proteins / Q000235:genetics*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D020928:Mitogen-Activated Protein Kinases / Q000032:analysis* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis*; D015398:Signal Transduction
chemical_list: D053823:Hedgehog Proteins; D012333:RNA, Messenger; C465570:Shh protein, mouse; D020928:Mitogen-Activated Protein Kinases